The enumeration of absolute levels of cells and their subsets in body fluids is of primary importance in determining the state of health of human beings and mammals in general. The primary analytical platform for performing such analyses is flow cytometry in which the specimen is either injected directly or after prior enrichment in rare cell analysis. Flow cytometry and similar complex analytical systems remain largely inaccessible for routine clinical use in resource-poor countries due to high instrument and reagents costs, lack of technical support, lack of robustness requiring frequent service, and the need for AC power. There is a clear need for simpler, more compact and less expensive systems also operable with emergency DC battery power and preferably exhibiting comparable performance characteristics.
In addition to the above-cited full sized flow cytometry systems available from Becton Dickinson and Beckman-Coulter, these vendors also sell scaled down less expensive versions, which still suffer from the other cited limitations. Similar limitations apply to the compact CyFlow® from Partec GmbH, (Munster, Germany) and to the Guava Personal Cytometer (Burlingame, Calif.). U.S. Pat. No. 6,097,485 (assigned to Integrated Wave Guides, Brookings, S. Dak.) discloses an ultra-miniature personal flow cytometer (pFCM) claimed to be of lower cost, but still exhibiting rather complex, electronic circuitry, optical designs, data reduction, all of which contribute to unacceptable complexity for a third world setting. All these systems use the flow concept, which obviously complicates the instrumental design. These scaled down versions of flow cytometry systems do not meet the clear need for a truly simple, compact, rugged, battery-operable and affordable cell analyzer.
Among the numerous clinical applications for a simple cell analyzer, counting of CD4 cells in HIV, granulocytes and platelets in patients treated with chemotherapy, and leukocytes in blood bags are most important. The current systems and methods for cell analysis have some significant disadvantages. They generally require sophisticated techniques, which involve the use of instruments that are expensive both in terms of initial cost and maintenance as well as requiring highly trained personnel. This makes the conventional systems unsuitable for use in laboratories of resource-poor countries. Therefore, a low-cost, easy-to-use method, for example, for CD4 cell enumeration is needed. Such a method may serve as a compact alternative to the current cell analysis systems that would be suitable for physician practices, bedside testing, or in open field settings.
HIV and AIDS are the leading cause of death in Africa and the fourth leading cause of death worldwide. In the countries most affected, life expectancy has declined by 10 years and infant death rates have doubled. In countries with the highest HIV prevalence, such as Botswana, South Africa, and Zimbabwe, the full impact of the epidemic has not yet been felt because those infected recently have not yet developed overt symptoms. Equally important is the effect of HIV deaths on families, social systems, and national growth and development. Young adults who contribute substantially to the countries' gross domestic product are most commonly affected. The most effective intervention therapy for persons infected with HIV is the use of a combination of antiretroviral agents. However, the high cost of these regimens and the infrastructure needed to monitor their use have put these medications beyond the reach of most HIV-infected persons. Although the price of these drugs has fallen, making treatment a possibility for a greater number of persons, infrastructures to support the effective use of these medications remain inadequate and need strengthening. To characterize disease in an individual for purposes of estimating prognosis and planning therapy, clinicians need to know how far the disease has progressed.
In HIV disease, currently this is most usefully indicated by the CD4 count. HIV infects the CD4 positive subset of T-lymphocytes, eventually leading to their depletion and the onset of the various opportunistic infections manifested in AIDS. During the course of HIV infection, the number of CD4+ T-lymphocytes drops from the normal value of about 500 to 1300 cells/μl to less than 200 cells/μl. The natural course of HIV in a typical untreated patient begins with a sharp rise of virus in the blood and a consequent drop in CD4+ T-cells. The immune system soon recovers to some extent, however, and keeps HIV levels fairly steady for several years. Eventually, though, the virus gains the upper hand. AIDS is diagnosed when the CD4+ T-cell level drops below 200 cells per cubic millimeter of blood or when opportunistic infections (reflecting failed immunity) arise, whichever happens first. CD4+ T-cell levels can thus be used to determine the progression of the HIV disease. Furthermore, routine monitoring of absolute CD4 counts provides important information about determining the effectiveness of therapy, as well as the response to therapy.
Based upon the demands, the following design criteria was established for using the present invention to detect and enumerate CD4 cells in HIV patients:                1. Enumeration should be possible from 100 to 2500 CD4+ T-cells/μl blood, but most critically in the 100-500 range. A count greater than 500 cells/□l is irrelevant. Also less than 200 cells/μl is the real critical level upon which clinical entry for therapy is advocated (see WHO treatment in resource poor settings). These latest guidelines stratify CD4 very simply and use hemoglobin (Hb) to clinically stratify patients.        2. Number of false positives (monocytes, other cells) below 10%. This is especially important where monocytes may be high e.g. co-infection with TB. TB is regarded as an AIDS defining illness below CD4 of 200/uL, and in the experience in South Africa cases, most of the CD4 counts are in the less than 200-400 range.        3. Cost price of hardware $1000 or less.        4. Maximum cost per test (including chemicals, antibodies etc.) $1 or lower. Flow cytometry using generic reagents costs less than $1-2 per test.        5. Minimal amount of laboratory handling required. This is an essential point for application in a resource-poor setting.        6. Use of disposables (cuvetes etc.) to avoid cleaning steps and for safety reasons. This does not mean that disposable systems only should be developed. In a very resource-poor setting, disposables may be beyond the reach, and a cleanable chamber might then be better. To prevent that “all” (disposables and blood) goes in the general garbage or down the drain, it may be a good idea to include a simple sterilizing system for disposables.        7. The overall system should be rugged and portable; have low power consumption (is battery-operated) and have an automatic data registration.        
The invention described herein meets the criteria above. The invention uses a CCD camera to image samples. Object detection algorithms are performed on the captured image to count the number of target entities present in a sample.
The prior art contains many computer-assisted microscopes. U.S. Pat. No. 5,018,209 teaches a computer driven microscope in which the user manually selects positive events while looking at an image. Obviously, this does not have a high enough throughput to be an effective analyzer, especially in remote settings.
In U.S. Pat. No. 5,287,272, an automated cytological specimen classification system and method is described. This system relies on a complicated neural network to process images of cells based on morphology. While very effective for classifying objects in images, it requires a large amount of computational resources. Furthermore, human input and subsequent analysis is still necessary. Other devices, such as those described in U.S. Pat. Nos. 5,073,857 and 5,077,806, use window sub-image pixel counting algorithms for image analysis by using predetermined thresholds.
Another set of instruments in the prior art is designed as bench top analyzers. In U.S. Pat. No. 5,073,857, pap smears are analyzed by a computer controlled microscope and camera and computer driven image analysis. In U.S. Pat. No. 6,221,607, an automated microscope is described for analyzing in situ hybridization events in a biological specimen.
The devices in the aforementioned prior art are designed to image slides. None are capable of detecting and enumerating a target population within a biological specimen as defined herein. Furthermore, none appear to be portable or high throughput devices. These instruments are designed to rely on a desktop computer to control the microscope and camera, and to perform image analysis algorithms. The present invention overcomes many of the difficulties that lie in the prior art.